A wide range of cell-surface proteins, including enzymes, coat proteins, surface antigens, and adhesion molecules, are attached to plasma membranes via glycosyl-phosphatidylinositol (GPI) anchors. GPI anchors are also proposed to function in protein targeting, transmembrane signaling, and in the uptake of small molecules (endocytosis). GPI anchors of plasma membrane proteins are present in eukaryotes from protozoa and fungi to vertebrates (Doering, T. L. et al. (1990) J. Biol. Chem. 265:611-614; McConville, M. J. et al. (1993) Biochem. J. 294:305-324).
GPI anchor precursor molecules are synthesized in the endoplasmic reticulum (ER) by the sequential addition of carbohydrate and other moieties to phosphatidylinositol (PI). The GPIs are transferred to the C-termini of proteins in the ER lumen. The GPI-anchored proteins are then transported to the cell surface via the secretory pathway.
The first step in GPI assembly is the transfer of N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to PI to form the intermediate GlcNAc-PI. In both yeast and mammals this process involves at least three proteins (Leidich, S. D. et al. (1994) J. Biol. Chem. 269:10193-10196; Hyman, R. (1988) Trends Genet. 4:5-8). The genes encoding three proteins involved in GlcNAc-PI formation in the yeast S. cerevisiae, GPI1, GPI2, and GPI3, have been cloned. The isolation of conditionally-lethal mutants of these three proteins has demonstrated that the first step in GPI assembly is essential for yeast viability (Leidich, et al. (1994), supra; Leidich, S. D. et al. (1995) J. Biol. Chem. 270:13029-13035). Human homologs of GPI1 and GPI3 have been cloned and expressed (Watanabe, R. et al. (1996) J. Biol. Chem. 271:26868-26875).
Paroxysmal nocturnal hemoglobinuria (PNH) is a disorder linked to defective GPI anchor biosynthesis. PNH is an acquired blood disorder which results from a somatic mutation in hematopoietic stem cells. Red blood cells arising from the PNH stem cells are highly sensitive to complement-mediated lysis and are prone to intravascular hemolysis. PNH may evolve to aplastic anemia or to acute leukemia. PNH cells are deficient in membrane surface GPI-anchored proteins due to defects in GPI biosynthesis (Hillmen, P. et al. (1993) Proc. Natl. Acad. Sci. USA 90:5272-5276).
GPI anchors play a role in sorting and directing proteins to distinct regions of the plasma membrane (reviewed in Turner, A. J. (1994) Essays Biochem. 28:113-127). GPI anchoring normally correlates with localization to the apical rather than the basolateral surface of polarized epithelial cell lines. In hippocampal neurons, the GPI-anchored Thy-1 glycoprotein is directed exclusively to axonal membranes. The GPI anchor may direct transport of the modified proteins to the apical cell surface by way of apical transport vesicles from the trans-Golgi network. A similar mechanism may operate in neuronal cells (Turner, supra).
GPI anchors also play a role in clathrin-independent endocytosis of small molecules into cells. GPI-anchored receptors for these small molecules cluster on the plasma membrane surface in specialized regions called the caveolae. These caveolae appear as flask-shaped invaginations in electron micrographs. Binding of ligand, for instance 5-methyltetrahydrofolate to the folate receptor, induces the caveolae to close, pinch off, and internalize the receptor and its ligand. Small molecule uptake is facilitated by GPI-anchored enzymes on the surface of the caveolae which hydrolyze peptides, nucleotides, and carbohydrates so that the digested products are concentrated in caveolae and internalized (Turner, supra).
Protozoan parasites cause widespread and debilitating diseases in humans and domestic livestock throughout the tropics. Examples of these diseases include malaria (caused by Plasmodium falciparum), African sleeping sickness and the cattle disease nagana (caused by Trypanosoma brucei), Chagas' disease (caused by Trypanosoma cruzi), and kala azar, espundia, and Oriental sore (caused by Leishmania sp.). There are no vaccines against these diseases, and most of the available drug treatments are toxic and/or ineffective. Recently, the emergence of drug resistant Plasmodium have placed malaria back into this category. The World Health Organization has identified the development of new and safer treatments for these diseases as a major priority.
Carbohydrate structures fixed into the parasite membrane by GPI membrane anchors play vital roles in the life cycles of these parasites. The use of GPI anchors is far more pronounced in parasites than in animal cells; in fact, GPI-anchored proteins dominate the molecular architecture of the parasite cell surface. In several cases, GPI-anchored proteins, such as the variant surface glycoprotein (VSG) of the African trypanosomes, or GPI-related glycolipids, such as the lipophosphoglycan (LPG) of Leishmania, are known to be essential for parasite survival and infectivity (Ferguson, M. A. et al. (1994) Parasitology 108: S45-54). Functional differences between some GPI-anchor biosynthetic enzymes of protozoan parasites compared to mammals have been noted (Guther, M. L. et al. (1994) J. Biol. Chem. 269:18694-18701).
Fungal infections are also major health problems, especially among immunocompromised individuals, such as those patients receiving immunosuppressive therapy to prevent transplant rejection or to treat neoplastic diseases, inflammation, or human immunodeficiency virus (HIV). Infectious agents which do not normally cause disease in immunocompetent hosts cause serious disease in those who lack a complete immune system. For example, pulmonary infections by fungi such as Histoplasma sp. and Coccidioides immitis may be fatal in immunocompromised individuals, young children, or elderly patients. Patients with diabetes mellitus or hematologic malignancy, or those receiving broad-spectrum antibiotics or high doses of adrenal corticosteroids, are especially susceptible to tissue invasion by Candida. Aspergillus is another widespread fungus which does not commonly cause disease except in immunocompromised patients.
Discovery of a new human GPI2 homolog and the polynucleotides which encode it satisfies a need in the art by providing new compositions useful in diagnosing, preventing, and treating disorders associated with GPI-anchored proteins. Knowledge and expression of sequences encoding human GPI2 homolog is also useful for developing therapeutic agents to prevent or treat diseases associated with fungal and parasitic infections.